This invention relates to apparatus and methods for the enumeration, examination, and manipulation of magnetically labeled particulate entities, and especially biological particles, such as cells.
A magnetic material or magnetic dipole will move in a magnetic field gradient in the direction of increasing magnetic field strength. Magnetic gradients employed in fluid separations are broadly divided into two categories. Internal magnetic gradients are formed by inducing magnetization in a susceptible material placed in the interior of a separation vessel. External gradients are formed by an externally positioned magnetic circuit.
In the case of a simple rectangular bar magnet, for example, field lines which form magnetic circuits conventionally move from North to South and are easily visualized with iron filings. From this familiar experiment in elementary physics it will be recalled that there is greater intensity of field lines nearest the poles. At the poles, the edges formed at the intersections of the sides and faces of the bar will display an even greater density or gradient. Thus, a steel ball placed near a bar magnet is first attracted to the nearest pole and next moves to the region of highest field strength, typically the closest edge. For magnetic circuits, any configuration which promotes increased or decreased density of field lines will generate a gradient. In opposing magnet designs, such as N-S-N-S quadrupole arrangements, opposing North poles or opposing South poles will have field lines such that in the center of such an arrangement there will be zero field. From the circuits that result from a North pole being opposite to each adjacent South pole, such arrangements generate radial magnetic gradients.
Internal high gradient magnetic separators have been employed for nearly 50 years for removing weakly magnetic materials from slurries such as in the kaolin industry, or for removing nanosized magnetic materials from solution. (See Kolm, Scientific American, November 1975). In an internal high gradient magnetic separator, a separation vessel is positioned in a uniform magnetic field. A ferromagnetic structure is positioned within the vessel in order to distort the magnetic field and to generate an xe2x80x9cinternalxe2x80x9d gradient in the field. Typically, magnetic grade stainless steel wool is packed in a column which is then placed in a uniform magnetic field which induces gradients on the steel wool as in U.S. Pat. No. 3,676,337 to Kolm. Gradients as high as 200 kGauss/cm are easily achieved. The magnitude of the field gradient in the vicinity of a wire is inversely related to the wire diameter. The spatial extent of the high gradient region is proportionally related to the diameter of the wire. As will be explained below, collection of magnetic material takes place along the sides of the wire, perpendicular to the applied magnet field lines, but not on the sides tangent to the applied field. In using such a system, material to be separated is passed through the resulting magnetic xe2x80x9cfilterxe2x80x9d. Then, the collected material is washed, and the vessel is moved to a position outside the field where magnetic materials are removed, allowing the collector to be reused.
Various attempts have been made to perform continuous (non-cycle) high gradient magnetic separation. Improvements include flow-through devices with fluctuating fields to separate magnetic material from non-magnetic material, as in U.S. Pat. No. 3,902,994 to Maxwell. Removable screens of ferromagnetic material are also well known in the art as in U.S. Pat. No. 4,209,394 to Kelland. Other flow-through devices are described in U.S. Pat. Nos. 4,261,815 and 4,663,029 to Kelland, U.S. Pat. No. 4,526,681 to Friedlaender, et al., and commonly owned U.S. patent application Ser. Nos. 08/424,271 and 08/482,652.
A method and apparatus for separating cells and other fragile particles is described by Graham, et al in U.S. Pat. No. 4,664,796. The apparatus contains a rectangular chamber within a cylinder. One pair of opposing sides of the chamber are made of non-magnetic material, while the other sides are made of magnetic material. The flow chamber is packed with a magnetically responsive interstitial separation matrix of steel wool. The material to be separated is passed through the chamber, which is positioned in a uniform magnetic field. During separation, the chamber is aligned in the magnetic field such that the magnetic sides of the chamber are parallel to the applied field lines, thus inducing a high gradient about the interstitial matrix in the chamber. When the chamber is in this position, magnetically labeled cells are attracted to the matrix and held thereon, while the non-magnetic components are eluted. The chamber is then rotated, so that the magnetic sides face magnets, which xe2x80x9cshuntsxe2x80x9d or xe2x80x9cshort-circuitsxe2x80x9d the magnetic field, reclines the gradients in the flow chamber, and allows the particles of interest to be removed by the shearing force of the fluid flow.
Other internal magnetic separation devices are known. Commonly owned U.S. Pat. No. 5,200,084 discloses the use of thin ferromagnetic wires to collect magnetically labeled cells from solution. U.S. Pat. No. 5,411,863 to Miltenyi discloses the use of coated steel wool, or other magnetically susceptible material to separate cells. U.S. patent application Ser. No. 08/424,271 by Liberti and Wang discloses an internal HGMS device useful for the immobilization, observation, and performance of sequential reactions involving cells.
Turning to the magnetic particles used in such collection devices, over the last twenty years, superparamagnetic materials have become the backbone of magnetic separations technology in a variety of healthcare and bioprocessing applications. Such materials, ranging in size from 25 nm to 100 xcexcm, are characterized in that they are only magnetic when placed in a magnetic field. Once the field is removed, they cease to be magnetic and can normally easily be dispersed into suspension. The basis for superparamagnetic behavior is that such materials contain magnetic cores smaller than 20-25 nm in diameter, which is estimated to be less than the size of a magnetic domain. A magnetic domain is the smallest volume in which a permanent magnetic dipole exists. Magnetically responsive particles can be formed about one or more such cores. The magnetic material of choice is magnetite, although other transition element oxides and mixtures thereof having appropriate particle size exhibit such superparamagnetic behavior.
Magnetic particles of the type described above have been used for various applications, particularly in health care, e.g. immunoassay, cell separation and molecular biology. Particles ranging from 2 xcexcm to 5 xcexcm are commercially available from Dynal. These particles are composed of spherical polymeric materials into which magnetic crystallites have been deposited. These particles because of their magnetite content and size, are readily separated in relatively low external gradients (0.5 to 2 kGauss/cm). Another similar class of materials are particles manufactured by Rhone Poulenc which typically are produced in the 0.75 xcexcm range. Because of their size, they separate more slowly than the Dynal beads in equivalent gradients. Another class of particulate magnetic material is available from Advanced Magnetics. These particles are basically clusters of magnetite crystals, about 1 xcexcm in size, which are coated with amino polymer silane to which bioreceptors can be coupled. These highly magnetic materials are easily separated in gradients as low as 0.5 kGauss/cm. Due to their size, both the Advanced Magnetics and Rhone Poulenc materials remain suspended in solution for hours at a time.
There is a class of magnetic particles which has been applied to bioseparations and which have characteristics which place them in a distinct category from those described above. These are nanosized colloids (see U.S. Pat. No. 4,452,773 to Molday; U.S. Pat. No. 4,795,698 to Owen, et al; U.S. Pat. No. 4,965,007 to Yudelson; U.S. Pat. No. 5,512,332 to Liberti and Piccoli and U.S. Pat. No. 5,597,531 to Liberti et al.; and U.S. patent application Ser. No. 08/482,448 of Liberti et al). They are typically composed of single crystal to multi-crystal agglomerates of magnetite coated with polymeric material which make them compatible with aqueous liquids. Individual crystals range in size from 8 to 15 nm. The coatings of these materials have sufficient interaction with solvent water to keep them permanently in a colloidal suspension. Typically, well coated particles below 150 nm will show no evidence of settling for as long as 6 months. These materials have substantially all the properties of ferrofluids.
Because of the particle size and strong interaction with solvent water, substantial magnetic gradients are required to separate ferrofluids. It had been customary in the literature to use steel wool column arrangements, such as described above, which generate 100-200 kGauss/cm gradients. However, it was subsequently observed that such materials form xe2x80x9cchainsxe2x80x9d (like beads on a string) in magnetic fields, thus allowing separation in gradient fields as low as 5 or 10 kGauss/cm. This observation led to development of separation devices using large gauge wires which generate relatively low gradients. Large gauge wires can be used to cause ferrofluids to produce uniform layers upon collection. By controlling amounts of ferrofluid in a system, a monolayer can be formed. Magnetically labeled cells can thus be made to form monolayers as described in commonly owned U.S. Pat. Nos. 5,186,827 and 5,466,574.
Analysis of the cellular composition of biological fluids is used in the diagnosis of a variety of diseases. Microscopic examination of cells smeared or deposited on slides and cytochemically stained has been the traditional method for cell analysis. Introduction of impedance based cell counters in the late 1950""s has led to a major advance in the accuracy of cell enumeration and cell differentiation. Since then various other technologies have been introduced for cell enumeration and differentiation such as Flowcytometry, Fluorescence Activated Cell Sorting, Quantitative Buffy Coat Analysis, Volumetric Capillary Cytometry, Laser Scanning Cytometry and various image analysis systems. Fluorescence based flowcytometry has dramatically improved the ability to discern different cell types in heterogeneous cell mixtures. This technique is commonly used, for example, to measure the absolute and relative number of cells in a specific subset of leukocytes in blood. In practice, a blood sample is drawn and incubated with a fluorescently labeled antibody specific for this subset. The sample is then diluted with a lysing buffer, optionally including a fixative solution, and the dilute sample is analyzed by flow cytometry. This procedure for analysis can be applied to many different cell surface antigens. Simultaneous assessment of multiple parameters of individual cells which pass the measurement orifice of a flow cytometer at a speed of up to 1,000 to 10,000 cells/sec is indeed a powerful technology. However, there are limitations on this technology, such as the inability to conveniently accommodate high cell concentrations (e.g., blood needs to be diluted), impracticality of the detection of infrequent or rare cells, and the inability to reexamine the cells of interest. In such situations, the time needed for the flow cytometer to analyze these samples becomes extremely long, thus decreasing the sample throughput. In addition, the settling of cells in the sample tube will occur during this time and require continuous mixing of the sample. To overcome these limitations, clinical samples to be analyzed are typically subjected to various enrichment techniques such as erythrocyte lysis, density separation, immunospecific selection or depletion of cell populations prior to analysis by flow cytometry.
Coated surfaces have been used for many research and clinical applications over the last several decades. Coated plates, reaction vessels, and tubes are well-known in the art. Coated surfaces have found particular use in immunoassays, and have seen wide usage since the 1960""s, starting with radioimmunoassays. Coated cup assays remain common in many of the clinical analyzers in laboratory use today, such as the VITROS ECI, Cyber-fluor, Delfia and the ES-300 systems. See also U.S. Pat. No. 4,376,110. Advantages of coated cup assays include that they provide a single, essentially homogeneous layer of analyte for analysis, which will withstand vigorous wash steps and result in low non-specific binding.
Commonly owned U.S. patent application Ser. No. 08/516,694 to Rao and Liberti relates to the use of a coated surface combined with a magnetic immunoassay. Magnetically collected material is immobilized on a coated surface through a specific binding pair reaction. The specific binding pair is borne upon the magnetic particle, which results in the non-reorientation of the magnetically collected material, when such particles are perturbed by sample removal or buffer addition and removal. The formation of a monolayer of magnetic material upon the coated surface is also facilitated, which reduces trapping of potentially signal-interfering substances. As a result, resuspension during washing or during signal readout is not required. Additionally, since the binding pair is adhered to the magnetic particle, all magnetic particles become bound to the coated surface, which prevents the loss of particles during washing, resulting in higher signal. However, in the separation of other biological material, such as cells, other concerns must be taken into account. Sample sizes are generally larger than in immunoassay, requiring larger volumes of magnetic material. Additionally, cells are much larger than the analytes in immunoassays, thus requiring larger amounts of magnetic particle reagents in order to convey the cell in the magnetic field. Increased amounts of such reagents are also required to drive the binding reaction, resulting in significant excesses of magnetic material. The sample volume reduction needed to concentrate the cells for analysis also results in an increase in the concentration of magnetic particle reagents. Such a large excess of material tends to obscure the microscopic examination of cells and inhibits their further analysis.
Other cytological techniques involve cell deposition on a slide for microscopic analysis. Sample preparation for these techniques include cell centrifuges, the Cytoshuttle, sorting of cells by Fluorescence Activated Cell Sorting (FACS), or other cytometric techniques by which target cells are separated after analysis/identification, such as B-D""s FACS Sort. Cell centrifuges are sold by several companies, including Shandon Lipshaw and StatSpin. In these systems, a centrifuge is used to deposit a cell sample on a microscope slide. However, drawbacks of the system include cell loss in the centrifugation step and the inability to selectively deposit target cells onto a slide. A pre-selection of cells is required in such cases. A pre-selection is also required for the Cytoshuttle, available from Cancer Diagnostics, which uses a filter to collect cells on special filter paper. The cells collected on the filter paper are then transferred to a microscope slide for analysis. Cell loss is also a problem with the Cytoshuttle.
Cell sorting additions to flow cytometers have been sold by Becton-Dickinson (B-D), Coulter, and Ortho. Shapiro""s Practical Flow Cytometry, (3rd ed. Wiley-Liss, NY, 1995) provides a comprehensive description of the theory behind this apparatus. Basically, there are two types of sorters, droplet and fluidic. The droplet sorters include FACSVantage (B-D), MoFlo (Cytomation) and EPICS (Coulter), which divide the fluid stream into individual charged droplets, some of which contain cells. Charged plates deflect the droplets into one or two streams from which they can be collected. In fluidic sorters, such as the FACS Sort (B-D), a mechanical arm is placed in the fluid stream. When a target cell passes, the arm is extended into the sample stream, capturing the fluid containing the target cell and then moving back to its original position in the fluid stream. Since the arm is placed in the fluid stream, a continuous flow of fluid is collected along with the target cell, resulting in a considerable dilution of the target cells.
The ability to deposit target cells in specific position for analysis has been described by Stovel and Sweet (J Hitochem and Cytochem, 27: 284-288 (1979)) and is commercially available as B-D""s Accurate Cell Deposition Unit (ACDU). The ACDU is an option added to a droplet sorter which allows sorting of cells into a microtiter well or onto a microscope slide. The slide is guided via computer control to capture target cells on a pre-defined region of the slide. Thus, one is able to sequester individual cells on a portion of a microscope slide, although it is difficult to maintain cell integrity and morphology. However, in the Stovel and Sweet system, the cells are sorted into individual xe2x80x9csplash circlesxe2x80x9d with a 270 micron diameter, making it relatively difficult to locate a single cell within this large diameter. Additionally, not all cells which fall within the sort gate of the scatterplot are actually deposited on the slide, and it is thus impossible to correlate the individual deposited cells to the cells in the sort gate. This problem has been overcome by adding an additional analysis point to determine if the gated cell was actually successfully sorted. This system has been used to index cells sorted into microtiter wells. See Terstappen et al., xe2x80x9cCharacterization of Human Primitive Hematopoietic Stem Cellsxe2x80x9d, presented at Joint International Workshop on Foetal and Neonatal Hematopoiesis and Mechanisms of Bone Marrow Failure, Apr. 3-6, 1995, Paris. Despite these features, droplet sorting is inherently more complex than fluidic sorting and such instruments could not practically be used in a clinical setting, even with highly trained technicians.
Fluidic sorters of the type described in U.S. Pat. No. 5,030,002 to North are relatively easy to maintain and operate, but they have their own set of drawbacks. The mechanical arm is positioned in the fluid stream and liquid is continuously collected by the arm. A target cell is caught when the arm moves into the sample stream. The sheath fluid rate thus determines the collection volume. For example, when the contents of a 500 xcexcl sample has to be sorted, it will take approximately 500 seconds (sample flow 1 xcexcl/sec.) and will produce a total volume of about 50 ml (sheath fluid rate of 18 ml/min. and catcher arm rate of 6 ml/min.). Irrespective of the number of captured cells, the volume will be approximately 50 ml.
From the foregoing discussion, it will be appreciated that a need exists for apparatus and methods which are capable of efficiently and effectively separating magnetically labeled particles, such as cells, from a fluid medium, including whole blood or sheath fluid, resulting in the capture of the cells of interest in a pre-determined pattern on a non-magnetic capture surface, such as a microscope cover slip.
The present invention provides apparatus and methods for the collection and immobilization of particulate entities, especially those of biological origin, such as cells.
The particulate entities are labeled with magnetically responsive particle and collected on a collection surface, followed by immobilization on the collection surface as a result of an interaction between the members of a specific binding pair, one member of which is uniformly or non-uniformly affixed to the collection surface, and the other member of which is associated with the particulate entity sought to be immobilized.
In a particularly preferred aspect of the invention, an apparatus is provided for immobilization of magnetically labelled particulate entities on a collection surface via binding between the members of a specific binding pair. In this aspect of the invention, the particulate entities comprise one member of the specific binding pair, with the other member being affixed to at least a portion of the collection surface. The preferred apparatus of the invention comprises magnetic means for providing a magnetic field, a collection surface disposed in the magnetic field generated by the magnetic means, and having affixed thereto the other member of said specific binding, as noted above, and ferromagnetic localization means operably associated with the collection surface for producing a magnetic field gradient having a defined pattern on the portion of the collection surface bearing the other specific binding pair member, whereby the magnetically labelled particulate entities are caused to adhere to the collection surface under the influence of the magnetic field gradient and to become immobilized on the collection surface when subjected to conditions promoting reaction between the specific binding pair members.
As will appear from the following description, the present invention offers a number of notable advantages over existing analytical techniques for separation and analysis of particulate analytes, e.g., cells or microbes, within test samples such as bodily fluids, culture fluids or samples from the environment, which may contain non-magnetic components. One distinct advantage of the present invention is maintenance of the target entities intact and/or viable upon separation to permit analysis, identification, or characterization of the target entities.